Transcript Transmission Electron Microscopy (TEM)

Advances in Bioscience Education
Summer Workshop
Immunolabeling for
Fluorescence and Electron Microscopy
June 27 - 29, 2006
Biological Electron Microscope Facility
Pacific Biosciences Research Center
University of Hawai’i at Manoa
Biological Electron Microscope Facility
 Pacific Biosciences Research Center, University of
Hawai’i at Manoa
 Instrumentation, service and training
 State-of-the-art instruments for biological
microscopy
 In operation since 1984
 Personnel:
 Dr. Richard D. Allen, Director
 Dr. Marilyn F. Dunlap, Manager
 Tina M. (Weatherby) Carvalho, M.S., Supervisor
Light and Electron Microscopy
 Light microscopy
 Glass lenses
 Source of illumination is usually light of visible
wavelengths
 Tungsten bulb
 Mercury vapor or Xenon lamp
 Laser
 Electron microscopy
 Electromagnetic lenses
 Source of illumination is electrons
 Hairpin tungsten filament (thermionic emission)
 Pointed tungsten crystal (cold cathode field emission)
 Lanthanum hexaboride
Epifluorescence Microscopy
 Olympus BX51
upright microscope
 Broad-band
epifluorescence
excitation and
detection
 DIC optics
 Optronics scientific
grade digital camera
Epifluorescence
Green photos courtesy Dr. Teena Michaels, KCC
Red photo courtesy Dr. Claude Jourdan-LeSaux
Common Fluorescence Applications
Localize/identify specific organelles
Detect live cells vs. dead cells, necrotic vs.
apoptotic cells
Determine cell membrane permeability
Localize antigen-specific molecules
Multiple labeling
Laser Scanning Confocal
Microscope
Olympus Fluoview
FV1000
Three colors + Transmitted simultaneously
Excitation with 405,
458, 488, 515, 543, and
633 nm lasers
Various emission filters
Optical sectioning
3-D reconstruction
Stereo views
Animations
Laser Scanning Confocal Microscopy
Drosophila eye
Photo courtesy of Gregg Meada & Dr.
Gert DeCouet, UHM
 Adjustable pinhole
aperture eliminates
out-of-focus glare
 Better resolution
 Serial optical
sections can be
collected from thick
specimens
 Live or fixed cell
and tissue imaging
Epifluorescence vs. Confocal
Sample courtesy Gregg Meada &
Dr. Gert DeCouet, UHM
Field Emission Scanning Electron
Microscopy (FESEM)
 Hitachi S-800 FESEM
 High magnification (40x
to 300,000x)
 High resolution (better
than 2 nm)
 Easy to learn
 Hi-res digital images
 Prep equipment:
critical point dryer,
sputter coater
SEM Images
Transmission Electron Microscopy
(TEM)
 Zeiss 10/A conventional
TEM
 Excellent for training
 Film only
LEO 912 Energy-Filtering TEM
 In-column energy filter
(electromagnetic prism)
 Ultrathin to 0.5 µm sections
 Contrast tuning
 Elemental analysis with
electron energy loss
spectroscopy (EELS)
 Elemental mapping with
electron spectrographic
imaging (ESI)
 Eucentric goniometer stage
 Digital images
Conventional TEM Micrographs
Skin
Bacteria in cell
Apoptosis
Chloroplast
Collagen
Virus in cell
Negative Staining
 Viruses, small
particles, proteins,
molecules
 No sectioning
 Same day results
EFTEM - Electron Spectrographic
Imaging (ESI) - elemental mapping
 Calcium in
mitochrondria
from ischemic
brain
 Iron in liver
EFTEM- Electron Energy Loss
Spectroscopy (EELS)
 EELS spectrum
Ultramicrotomy
 Ultrathin (60-90 nm)
sectioning of resinembedded specimens
 Several brands/models
available
 Cryoultramicrotomy
Cryotechniques
 Ultrarapid cryofixation
 Metal mirror impact
 Liquid propane plunge
 Freeze fracture with
Balzers 400T
 Cryosubstitution
 Cryoultramicrotomy –
Ultrathin frozen
sections (primarily for
antibody labeling)
Cryo Examples
 Freeze fracture,
deep-etch, rotary
shadow
 Cryosection/immunogold label
 Cryosubstitution
Image Manipulation and Analysis
 Soft Imaging System
analySIS professional
software
 EFTEM acquisition and
analysis
 Light Microscopy
 Images from other sources
 Particle counting and
analysis
 Feature extraction
 Image and results database
Immunolocalization
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LM
Fluor/confocal
TEM
SEM with
backscatter
detector
Approaches to Immunolabeling
Direct Method: Primary antibody contains
label
Indirect Method: Primary antibody
followed by labeled secondary antibody
Amplified Method: Methods to add more
reporter to labeled site
Protein A Method: May be used as
secondary reagent instead of antibody
Direct Labeling Method
Labeled primary
antibody reacts
directly with the
antigen in the
histological or
cytological
preparation
Two-step Indirect Method
Fluorescentconjugated
secondary
antibody attaches
to primary antibody
that is bound to
antigen
Amplified Method
If the antibody
reporter signal is
weak, the signal
can be amplified by
several methods,
e.g., streptavidinbiotin complex
Double-labeling Method
 Use primary antibodies
derived from different
animals (e.g., one
mouse antibody and
one rabbit antibody)
 Then use two
secondary antibodies
conjugated with
reporters that can be
distinguished from one
another
Immunolabeling for Transmission
Electron Microscopy
 Normally do Two-Step
Method
 Primary antibody
applied followed by
colloidal gold-labeled
secondary antibody
 May also be enhanced
with silver
 Can also do for LM
Preparation of Biological Specimens
for Immunolabeling
 The goal is to preserve tissue as closely as
possible to its natural state while at the same time
maintaining the ability of the antigen to react with
the antibody
 Chemical fixation of whole mounts prior to
labeling for LM
 Chemical fixation, dehydration, and embedment in
paraffin or resin for sectioning for LM or TEM
 Chemical fixation for cryosections for LM
 Cryofixation for LM or TEM
Chemical Fixation
 Antigenic sites are easily denatured or masked during
chemical fixation
 Glutaraldehyde gives good fixation but may mask
antigens, plus it is fluorescent
 Paraformaldehyde often better choice, but results in
poor morphology , especially for electron microscopy
 May use e.g., 4% paraformaldehyde with 0.5%
glutaraldehyde as a good compromise
Preembedding or Postembedding
Labeling
 May use preembedding labeling for surface
antigens or for permeabilized cells
 The advantage is that antigenicity is more likely
preserved
 Postembedding labeling is performed on
sectioned tissue, on grids, allowing access to
internal antigens
 Antigenicity probably partially compromised by
embedding
Steps in Labeling of Sections
 Chemical fixation
 Dehydration, infiltration, embedding and
sectioning
 Optional etching of embedment, permeabilization
 Blocking
 Incubation with primary antibody
 Washing
 Incubation with secondary antibody congugated
with reporter (fluorescent probe, colloidal gold)
 Washing, optional counterstaining
 Mount and view
Controls! Controls! Controls!
Omit primary antibody
Irrelevant primary antibody
Pre-immune serum
Perform positive control
Check for autofluorescence
Check for non-specific labeling
Dilution series
Dilutions are Important
 Typically should do an
extensive dilution series to
determine best
concentration of both
primary and secondary
antibodies
 This shows an antibody at
concentrations of 1:100 and
1:2000
Know Your Artifacts
 And use them to your advantage!
 Green is label; orange-red is
autofluorescence
 Acts as counterstain
Autofluorescence
 Need to select label
that will be readily
distinguished from
autofluorescence
 Several techniques
to quench
autofluorescence
What is a Microscope?
 A tool that magnifies and improves resolution of
the components of a structure
 Has three components: one or more sources of
illumination, a magnifying system, and one or
more detectors
 Light microscopes use a beam of light for
illumination and include fluorescence and
confocal microscopes
 Electron microscopes use electrons as a source
of illumination and include transmission and
scanning electron microscopes
Light and Electron Microscopes
 Lenses are
used to control
a beam of
illumination,
magnify, and
direct an image
to a detector
Light Microscopes
Objective Lenses
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Objective lens choice is important!
Not all objective lenses are created equal
The more correction a lens has, the less transmission
Resolution is dictated by Numerical Aperture (NA)
Talk to your microscope company representative
Light Microscopes - Resolution
 Resolution depends
on the light gathering
of the objective, which
depends on the NA,
and on the light path,
which includes the
slide, sample,
mounting medium,
coverslip, and air or
immersion oil
Light Path in Fluorescence
 Light delivered
through excitation
filter and then
objective lens to
specimen where it
is absorbed;
emitted light goes
back through
objective lens
through barrier
filter and emission
filter and then to
detector.
Fluorescence Microscopes
 Illumination light path is
the same as the sampling
light path
 Need to maximize the light
throughput in both
directions – no more than
22% of light will be
detected on a good day
 Need to match refractive
indices (RI)
 Use the best optics with the
fewest elements
Optical Choices for Fluorescence
 Minimize the number of lens elements to increase light
throughput, but correct for spherical aberration
 Optimize magnification and NA; best choice often a 60X
1.4NA plan objective
 Only use magnification required to collect the information
needed
 Use a mercury lamp for normal work and a xenon lamp for
quantitative studies
Kohler Illumination
 Kohler illumination is essential for good
transmitted light contrast
 Focus slide
 Close field diaphragm
 Focus diaphragm in field by adjusting condenser
height
 Center diaphragm in field
 Open diaphragm to fill field and recheck
centration
 Adjust iris diaphragm (on condenser) to taste
(affects contrast and depth of focus)
Elements of Fluorescence Microscope
 Light source
 Mercury vapor
 Xenon
 Laser
 Optical lenses
 Optical filters
 Detection system
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Eye
Film camera
Digital camera
Photomultiplier tube (PMT)
Fluorescence
 Photons of a
certain energy
excite the
fluorochrome,
raising it to a
higher energy
state, and as it falls
back to it’s original
state it releases
energy in the form
of a photon of
lower energy than
the excitation
energy.
Fluorescence
 Fluorochromes are
excited by specific
wavelengths of light
and emit specific
wavelengths of a
lower energy
(longer wavelength)
Filter Cubes for Fluorescence
 Filter cubes
generally have an
excitation filter, a
dichroic element,
and an emission
filter
 The elements of
a cube are
selected for the
excitation and
fluorescence
detection desired
Classification of Filters
Long pass – passes longer wavelengths
Short pass – passes shorter wavelengths
Band pass – passes defined wavelengths
Dichromatic mirror – transmits long
wavelengths, reflects shorter wavelengths
Choose Fluorochrome/Filter Combos
Spectral Characteristics of Probes
 Omega Filters Curv-o-Matic
 http://www.omegafilters.com/front/curvomatic/spectra.php
 Other filter and microscope companies
Ideal Fluorochrome
 Small size – must get into cell
 High absorption maximum – sensitive to excitation
 Narrow absorption spectrum – excited by a narrow
wavelength
 High quantum efficiency – likely to fluoresce
 Narrow emission spectrum – so you can find it
specifically
 Large Stoke’s shift – emission curve far enough away
from excitation curve to minimize bleedthrough
Types of Fluorochromes
Simple dyes
 Acridine orange, DAPI, Propridium iodide, Lucifer yellow
Physiological probes
 Calcium green, Rhodamine 123, Fluorescein diacetate
Specific probes
 Phalloidin, Lectins, GFP, Primary and secondary antibodies
Laser Scanning Confocal Microscopy
 Fluorescence technique
 Uses laser light for excitation
 Improves image resolution over conventional
fluorescence techniques
 Optically removes out-of-focus light and detects
only signal from focal plane
 Can construct an in-focus image of considerable
depth from a stack of images taken from different
focal planes of a thick specimen
 Can then make a 3-D image that can be tilted,
rotated, and sliced
Principal Light Pathway in
Confocal Microscopy
 Laser light is scanned pixel
by pixel across the sample
through the objective lens
 Fluorescent light is reflected
back through the objective
and filters (dichroic mirrors)
 Adjustable pinhole apertures
for PMTs eliminate out-offocus flare
 Image is detected by
photomultiplier(s) and
digitized on computer
Compressed Z-stack Image
 3-D reconstruction
Tilt and rotate
 Stereo projection
 Animation
 Montage
 Image
enhancement
Photo courtesy Dr. Alex Stokes, Queens Medical Center
Confocal Movies
Photo courtesy Dr. Alex Stokes, Queen’s Medical Center
Confocal Projects
 Investigation of Wnt pathways in sea urchin
gastrulation (Dr. Christine Byrum/Dr. Athula Wikramanayake)
 Localization of transmembrane proteins in airway
smooth muscle cells (Dr. Lynn Iwamoto, Kapiolani)
 GFP in drosophila (Gregg Meada/Dr. Gert deCouet)
 Neurohormones (Dr. Ian Cooke/Toni Hsu)
 IL-10 receptors of lung fibroblasts (Dr. Claude JourdanLeSaux)
 Aggregation of acetylcholine receptors in muscle
cells (Drs. Jes Stollberg, UHM, and Michael Canute, HPU)
Differential Interference Contrast
(Nomarski)
Digital Imaging
Digital advantages include sensitivity,
speed, quantitation, feature extraction and
image analysis
CCD cameras - High resolution, slow
Video cameras – Low resolution, fast
Photomultiplier tubes (PMTs) – point
recorders, used for confocal
Digital Cameras
 Need enough sensitivity for signal you want to
detect
 Need enough speed for event you want to detect
 Need enough grayscales – 8 bits for
documentation, 12 bits for quantitation
 Need enough resolution - the number of of pixels
must be sufficient to distinguish features of
interest, but too many pixels is a waste of data
space
 Color is simply three black and white images
combined and useful primarily for image
processing
Optronics MacroFire Digital Camera
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Extremely sensitive
2048 x 2048 pixels
Millisecond exposures
Firewire
Fits on both Olympus
compound and stereo
zoom microscopes
 Suitable for BF, DF, and
Fluorescence
 Also Optronics MagnaFire
SP 1280 x 1024 pixels and
Nikon Coolpix cameras
TEM
 Transmission Electron
Microscope
 Illumination source is
beam of electrons from
tungsten wire
 Electromagnetic lenses
perform same function
as glass lenses in LM
 Higher resolution and
higher magnification of
thin specimens
Specimen Preparation for TEM
 Chemical fixation with buffered glutaraldehyde
 Or 4% paraformaldehyde with >1% glutaraldehyde
 Postfixation with osmium tetroxide
 Or not, or with subsequent removal from sections
 Dehydration and infiltration with liquid epoxy or
acrylic resin
 Polymerization of hard blocks by heat or UV
 Ultramicrotomy – 60-80nm sections
 Labeling and/or staining
 View with TEM
Colloidal Gold Immunolabeling for
TEM
Colloidal gold of defined sizes, e.g., 5 nm,
10 nm, 20 nm, easily conjugated to
antibodies
Results in small, round, electron-dense
label easily detected with EM
Can be enhanced after labeling to enlarge
size for LM or EM
Colloidal Gold in TEM
Colloidal Gold in TEM
Double Immunogold Labeling of
Negatively Stained Specimens
 Bacterial pili
serotypes dried
onto grid and
sequentially
labeled with
primary antibody,
then Protein-A5nm-gold and
Protein-A-15-nmgold before
negative staining
TEM Grids
 TEM grids are 3
mm supports of
various meshes
 You will handle
them by the
edges with fine
forceps
Colloidal Gold in SEM
 Gold particles are often
difficult to see against
the membrane with
secondary electron
detection
 Gold particles show up
brighter with
backscattered electron
detection
Preparation of Images for
Publication
Microscopy –
Images are your
data!
Adjustment and
labeling of images
for figure plates
with Adobe
Photoshop
How to Contact the BEMF
 Location: Snyder Hall 118 – University of Hawai’i at Manoa
 Phone: 808 956-6251
 FAX: 808 956-5043
 URL: http://www.pbrc.hawaii.edu/bemf
 E-mail: [email protected]
[email protected]
Acknowledgments
We thank all of the researchers who agreed
to let us use their images for this
presentation
Microscopy & Microanalysis 2005
 July 31 - August 4, 2005
 Hawaii Convention Center
 Over 1100 talks and posters
 Huge trade show featuring the latest in
microscopes and related instrumentation,
software, and support
 Pre-meeting workshops
 http://mm2005.microscopy.org